Nanoprogrammed Cross-Kingdom Communication Between Living Microorganisms

The engineering of chemical communication at the micro/nanoscale is a key emergent topic in micro/nanotechnology, synthetic biology, and related areas. However, the field is still in its infancy; previous advances, although scarce, have mainly focused on communication between abiotic micro/nanosystems or between microvesicles and living cells. Here, we have implemented a nanoprogrammed cross-kingdom communication involving two different microorganisms and tailor-made nanodevices acting as “nanotranslators”. Information flows from the sender cells (bacteria) to the nanodevice and from the nanodevice to receiver cells (yeasts) in a hierarchical way, allowing communication between two microorganisms that otherwise would not interact.

of nano/microparticles and living systems with advanced functions.
In this context, we present, as a proof-of-concept, to the best of our knowledge the first realization of a programmed crosskingdom communication involving two species of living cells enabled by tailor-made nanoparticles. In the first place, the engineered scheme comprises communication from the first type of cells to the nanoparticles in response to an external stimulus. Subsequently, the nanoparticles decode the received chemical message and emit a new message detected by the second type of cells which trigger a second response. The overall network can be described as living-to-abiotic-to-living cascade-like communication in which an abiotic nanodevice acts as "nanotranslator" allowing communication between two cells from different kingdoms that otherwise would not interact. In particular, we employed Escherichia coli (prokaryotic cells, bacteria kingdom) and Saccharomyces cerevisiae (eukaryotic cells, fungi kingdom) as model microorganisms. The "nanotranslator" consists of mesoporous silica nanoparticles loaded with a molecular messenger (phleomycin) and capped with a glucose oxidase (GOx)-based responsive gatekeeper. As illustrated in Scheme 1C, communication is triggered in the presence of lactose (input) which is sensed and hydrolyzed by E. coli cells (β-galactosidase-expressing, vide infra) into glucose and galactose. Glucose (first chemical messenger) is then detected by glucose oxidase (GOx) on the abiotic nanodevice, inducing the uncapping of the pH-sensitive gatekeeper and resulting in the release of phleomycin (second chemical messenger). Finally, in response to phleomycin S. cerevisiae yeast cells activate a genetic cascade that leads to green fluorescent protein (GFP) 35 expression and the subsequent production a fluorescence signal as the output of the communication network.
Interaction between species in our proposed system is carried out through an aqueous medium by means of chemical communication channels as both microorganisms have cell walls composed of proteins, lipids, and polysaccharides that avoid the internalization of nanoparticles unless specific permeability treatments are applied. 36,37 The engineered bacteria used in our studies (E. coli DH5α) carries a plasmid (pTZ57R) encoding lacZ (β-galactosidase production) and ampicillin resistance. The budding yeast strain employed expresses GFP upon exposure to DNA-damaging agents since its transcription is controlled by the RNR3 promoter. 38 Scheme 1. Representation of the Reported Nanoprogrammed Chemical Communication Paradigm between Microorganisms from Different Kingdoms a a (A) E. coli (β-galactosidase-expressing) bacterium cells do not communicate with S. cerevisiae yeast cells under normal conditions. (B) Tailormade mesoporous nanoparticles (loaded with phleomycin and capped with a GOx-based responsive gatekeeper) are added to enable communication. (C) Communication steps: bacterium cells convert lactose into glucose and galactose; glucose (first chemical messenger) is detected by the nanodevice inducing delivery of the entrapped phleomycin (second chemical messenger); finally, the receiver yeast cells sense phleomycin and respond by activating expression of GFP. Accordingly, GFP fluorescence signal is triggered in the presence of a genotoxin such as phleomycin. The "nanotranslator" is based on mesoporous silica nanoparticles due to the advantageous properties they have such as their chemical stability, large loading capacity and the great variety of cargoes which may be entrapped in their pores. Moreover, their surface can be decorated with a wide range of targeting groups, gatekeepers and enzymes showing a stimuli-responsive nature with tailor-made properties for versatile integration in communication scenarios. 39 In particular, our nanocarrier is based on mesoporous silica nanoparticles functionalized with benzimidazole (Bz) units on the external surface and capped by the formation of an inclusion complex with glucose oxidasemodified β-cyclodextrin (GOx-CD). This pH-sensitive supramolecular gatekeeper disassembles when glucose is present in the surroundings as the enzyme units produce gluconic acid inducing a local drop of pH and causing the protonation of benzimidazole moieties (pK a = 5.55); 40 the disruption of the benzimidazole:β-cyclodextrin complex leads to the uncapping of the pores and the delivery of the entrapped cargo.
To start with, we synthesized and characterized the sensingactuating nanoparticles (see Supporting Information for details). We first prepared GOx-functionalized nanoparticles loaded with a fluorescent dye ([Ru(bpy) 3 ]Cl 2 ) as model cargo. Indeed, the resulting nanoparticles had a spherical shape, a size of around 100 nm and a pore network as observed by transmission electron microscopy (Figures 1 and SI-1). In addition, powder X-ray diffraction, N 2 adsorption−desorption isotherms, dynamic light scattering, elemental analysis, enzymatic assays, and TEM-EDX were used to complete their characterization (Figure SI-2 to SI-6). Then, we tested the ability of the nanodevice to autonomously deliver the entrapped cargo upon exposure to glucose. To do so, we brought dye-loaded GOx-capped nanoparticles (NP GOx-Dye ) in aqueous solution (1 mg·mL −1 ) at pH 7.5 and monitored cargo delivery in the presence and absence of glucose by measuring the fluorescent signal of the released dye. A clear release was observed in the presence of glucose due to the opening of the GOx-CD-Bz gatekeeper; whereas in contrast, cargo delivery was insignificant in the absence of glucose ( Figure SI-7). Moreover, the specificity of the nanodevice was verified by confirming that cargo delivery was not observed in the presence of other saccharides, such as fructose, galactose, lactose, and sucrose ( Figure SI-9). After confirming the programmed sensing-actuating behavior, we prepared similar nanoparticles loaded with phleomycin (NP GOx-Phl ) that would have a receiver−sender role and enable the full communication shown in Scheme 1. We also confirmed that NP GOx-Phl was able to retain phleomycin and deliver it on-command in the presence of glucose ( Figure SI-8).
As a next step and envisaging the final designed communication system (Scheme 1C), we then checked the response of the selected microorganisms to their corresponding stimulus. First, for assessing the ability of engineered E. coli cells to process lactose, β-galactosidase expression was confirmed by qualitative and quantitative enzyme activity assays by means of X-Gal staining and o-nitrophenyl-β-Dgalactopyranoside hydrolysis in aqueous medium (determined β-galactosidase activity = 8.0 mU·mL −1 , culture OD = 0.5; see SI Section 13). Moreover, to test the response of yeast cells to phleomycin (chemical message), positive and negative control experiments were carried out by adding or not free phleomycin to yeast culture (at mid log exponential growth phase), that was further incubated for 3 h in the presence of E. coli. When coincubated ( Figure 1B Next, we set out to validate the first linear communication pathway of the network, that is, communication between bacteria (acting as sender) and the nanodevice NP GOx-Dye (acting as receiver). With this aim, we conducted a series of delivery studies in which E. coli bacterium cells (4 × 10 9 cells· mL −1 ) and NP GOx-Dye (1 mg·mL −1 ) were combined in aqueous solution (pH 7.5) in the absence or presence of lactose (2%, as trigger of the communication). As additional control, dye release from NP GOx-Dye in the absence of bacteria and the presence of lactose was also monitored. As plotted in Figure 2, a steady increase in cargo delivery ([Ru(bpy) 3 ]Cl 2 ) was   3 ]Cl 2 ) in aqueous solution at pH 7.5 containing NP GOx-Dye and bacteria in the absence (b, red curve) and presence (a, blue curve) of lactose (2%). As additional control, release from NP GOx-Dye in the presence of lactose and absence of bacteria was also monitored (c, black curve). Error bars correspond to the s.d. from three independent experiments. Nano Letters pubs.acs.org/NanoLett Letter observed in the complete combination (lactose + bacteria + nanoparticle), whereas no substantial dye release was observed either in the absence of lactose (bacteria + nanoparticle, red curve) or in the presence of lactose and absence of bacteria (lactose + nanoparticle, black curve) (see Table 1). Altogether, this corroborates the establishment of a linear communication model: bacteria are able to hydrolyze lactose (input) and catalyze the formation of glucose, which is sensed by the GOxcapped nanodevice with the subsequent cargo delivery. In the absence of bacteria, the nanodevice is insensitive to lactose as this disaccharide is not recognized by the GOx enzyme.
In our subsequent set of experiments, we tested the second linear communication pathway, that is, information transmission from the nanodevice to yeast cells. To do so, yeast cells (1.5 × 10 8 cells·mL −1 ) were incubated with phleomycinloaded GOx-capped nanoparticles (NP GOx-Phl ) in aqueous medium at pH 7.5 containing glucose (2%). As a control, we additionally prepared phleomycin-loaded nanoparticles lacking the GOx enzyme, yet capped with β-cyclodextrin (NP Phl ), and incubated them with yeast cells under the same conditions. After 3 h of incubation, induction of GFP expression was assessed by confocal fluorescence microscopy. As shown in Figure 3, the micrographs revealed a clearly higher fluorescent signal when yeast cells were incubated with NP GOx-Phl (panel a), as compared to nonfunctional NP Phl (panel b, lacking the enzyme). Quantification of the corresponding images (using ImageJ) revealed an about 5-fold increase in fluorescence upon incubation with functional NP GOx-Phl , as compared to control NP Phl . In order to address why certain (yet relatively low) GFP emission was observed in the negative control, we performed additional control experiments: (i) with no nanoparticles added but with glucose and (ii) with no nanoparticles but with glucose and the phleomycin equivalent corresponding to the determined background leakage ( Figure SI-13). Both of these additional controls showed a low GFP emission similar to the control with nonfunctional NP Phl ; thus, these experiments suggest that yeast cells exhibit certain background GFP expression under control conditions, yet GFP expression is considerably enhanced upon communication with the functional nanoparticles. Altogether, this confirms the ability of NP GOx-Phl to recognize glucose in the medium and deliver the phleomycin cargo (messenger) that triggers GFP expression in yeast cells. In nanoparticles lacking the GOx enzyme, the communication is disrupted.
After validating both linear communication pathways separately, we then constructed the complete nanoprogrammed cross-kingdom communication system. As depicted in Scheme 1, this involves a concatenated flow of information from the bacterium cells to the "nanotranslator" and subsequently to the yeast cells. To setup these experiments, yeast and bacteria were inoculated individually in fresh YPD medium and incubated until reaching mid log exponential phase. Then, both microorganisms were brought together in YPD medium (glucose-free, supplemented with fructose) and mixed with an aqueous solution at pH 7.5 of NP GOx-Phl (50 μg· mL −1 ). Then, 2% of lactose (input of the communication) was added. As control, parallel experiments were carried out with nanoparticles NP Phl (phleomycin-loaded β-cyclodextrin-capped nanoparticles lacking the GOx enzyme). Confocal fluorescence microscope images (Figure 4 and Figure SI-14) showed GFPassociated fluorescence when the "nanotranslator" NP GOx-Phl was present, whereas the fluorescent signal was significantly lower when the uncomplete nanoparticles NP Phl were employed. Quantification of GFP-associated fluorescence intensity from three independent experiments (Figure 4e) revealed more than a 4-fold emission increase in the presence of NP GOx-Phl , as compared to the control (i.e., NP Phl ). As additional control experiments to rule out any potential side interaction, we also prepared unloaded GOx-functionalized nanoparticles (NP GOx ) and unloaded nanoparticles also lacking GOx (NP Control ). As expected, significantly lower GFP expression was observed in confocal fluorescence microscopy studies in the same conditions when using NP GOx or NP Control , Presence or absence of input (lactose), bacteria and nanodevice is represented by + and − , respectively, whereas response refers to significant (+) or negligible (% <20%) (−) cargo delivery. Nano Letters pubs.acs.org/NanoLett Letter indicating that there is not chemical information flow when the nanoparticles did not contain cargo or/and enzyme. In addition, experiments in which bacteria and yeast cells were incubated in the absence of nanoparticles (see (−) in Figure  4e) showed similar GFP intensity levels as with control nanoparticles, which can be attributed to certain background expression in agreement with previous studies (see Table 2). 38 Furthermore, we determined the viability of yeast cells after conducting communication experiments based on the quantification of colony formation units (CFUs) after incubation for 24 h; no reduction in cell viability was observed when using nonfunctional NP Phl (lacking the enzyme). In contrast, a remarkable reduction in CFU counts was observed when functional NP GOx-Phl was added, which is ascribed to the genotoxic action of the released phleomycin ( Figure SI-16). These experiments demonstrate the hierarchical cross-kingdom communication of bacterium cells with yeasts through the use of an abiotic "nanotranslator" involving the directional exchange of two chemical messengers (glucose and phleomycin). The behavior of this communication network can be expressed in a Boolean logic table of five elements (i.e., the triggering input (lactose), the first microorganism (bacteria), the GOx enzyme on the nanodevice, the phleomycin cargo, and the receiver microorganism (yeast)). Among 32 possible entries (Table SI-4), only the complete system bacteria-NP GOx-Phl -yeast leads to effective cross-kingdom communication.
As an interesting (and so-far underexplored) aspect, spatial information transmission and propagation of sequential actions should be considered when designing chemical communication networks between micro/nanosystems. In an additional set of experiments, we employed microfluidic channels to control the relative spatial location of each communicating entity (bacteria−nanoparticles−yeast). As depicted in Figure 5, the experimental setup consisted of two reservoirs (60 μL) located  Table 2). Several fields of view of each condition were analyzed obtaining similar results. Data represent mean ± s.e.m. from thee independent experiments (*p < 0.001).
Corresponding with a−d micrographs and quantification in Figure 4. Overall, the engineered cross-kingdom communication cascade requires the exchange of two chemical messengers and the resulting production of a reporter protein. To better understand the dynamics of our multicomponent system, we decided to compare the relative signals of the two messengers and output signal at the same time scales. Similar communication experiments to as described above in bacteria−nanodevice−yeast mixtures were performed stopping the experiment at different times, that is, at 60, 120, and 180 min ( Figure SI-18). As depicted in Figure 6, a relatively low GFP signal was observed after 60 min incubation, which strongly increased at 120 min almost reaching saturation (∼96%). Moreover, no free glucose was detected in the mixture (using a commercial detection kit) at the scheduled times which suggested full consumption of glucose by the nanoparticles. Indeed, spectrophotometric assays (see SI for details) revealed that the rate of glucose production by bacteria (0.0034 μmol min −1 ) is lower than the rate of glucose consumption by the nanoparticles (0.084 μmol min −1 ). This also correlates with the fact that cargo release is slower in the linear lactose-triggered bacteria−nanoparticle communication experiments (Figure 3) as compared to when the nanoparticles are exposed to an equivalent concentration of glucose ( Figure  SI-8). In the absence of nanoparticles, we determined that the amount of substrate transformed by bacteria follows a linear trend ( Figure SI-10), as expected for first-order enzymatic reactions, which can be correlated to the relative signal corresponding to glucose production as showed in Figure 6. For the cargo release, we extracted the relative signal showed in Figure 6 by employing dye-loaded nanoparticles as previously described. Interestingly, at these time points signal 1 (generated glucose) and signal 2 (cargo release) followed a linear relationship ( Figure SI-19a), which could be potentially attributed to a coupling between the two signaling processes with glucose generation by bacteria being the limiting step. In contrast, comparison of these data also revealed that signal 3  Nano Letters pubs.acs.org/NanoLett Letter (GFP intensity) reached saturation faster than signal 2 (cargo release), which indicates effective activation of yeast cells once a certain partial release of cargo (∼75% at 120 min) is reached ( Figure SI-19b).
In summary, we report herein the nanoprogramming of cross-kingdom communication between living microorganisms, which involves two different cells and tailor-made nanoparticles acting as "nanotranslators". In our proof-of-concept system, molecular information from the environment (lactose) is processed by β-galactosidase-expressing E. coli bacteria and transformed into a chemical signal (glucose). Glucose is detected by the nanoparticles; subsequently, the nanoparticles translate the chemical message "glucose" to the chemical messenger "phleomycin" which is understandable for the receiver microorganism (S. cerevisiae). In response to phleomycin, S. cerevisiae yeast cells activate a genetic cascade that leads to green fluorescent protein expression as the output of the communication. The whole network can be described as two hierarchically concatenated linear communication pathways, that is, bacteria−nanodevice and nanodevice−yeast, which are independently validated. Cross-kingdom communication is demonstrated herein with functional nanoparticles that exhibited a double receiver-sender role, while communication is disrupted when the nanoparticles are incomplete.
This contribution is, as far as we know, the first realization of engineered cross-kingdom cellular communication mediated by nanoparticles and illustrates the potential to design chemical communication pathways at the micro/nanoscale involving several living and abiotic micro/nanosystems. The topic of chemical communication is still in its infancy and proof-of-concept demonstrations are a first necessary step toward the realization of future applications in fields such as biomedicine, microbiology and biotechnology. Whereas we based most of our experiments in standard well-established methods, the development of future applications will require more advanced methodologies to enable monitorization of chemical communication processes in complex settings such as biological tissues.
With development of "nanotranslators" that enable crosskingdom communication a wide range of applications can be envisioned. For instance, we might communicate messages that instruct cells to halt physiological processes or initiate protective behaviors; designing particles that can enable plants and fungi talk to each other could help us develop new ways to protect plants; while repurposing the finely honed language that some pathogens or cancer cells use to turn off the immune system may be a way to design new treatments for difficult-totreat diseases. Potentially, nanoparticles could be engineered as "nanokillers" to program the death of certain cells using chemical communication pathways, in fact, we observed the inhibition of yeast proliferation when the communication cascade is established, which as is an interesting area for further research. Ultimately, we envision that the cross-kingdom cellular communication enabled by nanoparticles will provide new therapeutic and diagnostic methods, biotechnological tools, ways to tune cellular behavior, and contribute to further increase our understanding of biological processes.